We are attempting, at the molecular level, to understand the regulation of differentiation in Bacillus subtilis by cloning regions of the B. subtilis chromosome containing sporulation markers. We will continue our efforts to clone these in E. coli using the lysogenic phage phi 105. The closest sporulation marker to the phi 105 attachment site is the SPO IV F locus, definitely a series of late block mutants. The next region contains the Spo 05 mutations, or early blocked mutants. Initial interest will be in the Spo 06 mutations in a study of the intial events of sporulation. The cloned DNA from the Spo IV F regions can be used in a comparative study, looking for changes in the regulation and expression of these regions. For our second approach we are requesting permission to clone B. subtilis genes in B. subtilis. With this system we plan to transform pIM13 into rec E containing auxotrophic hosts. By isolating specific gene fragments from the quadrants of the genetic a mini collection of marked plasmids should become available. We anticipate that the following problems will require detailed study: 1. the transformation of ciruclar versus linear DNA; 2. restriction and modification of the plasmid in new hosts; and 3. amplification of the plasmid.